Advances in Biology Laboratory Education Publication of the Association for Biology Laboratory Education Volume 41, Article 17, 2020 Using the Novel Dipstick DNA Extraction Technique in a Biological Barcoding Lab Christof Stumpf, Susan Bowers, and Nathan Sammons Louisiana State University at Alexandria, Department of Biological Sciences, 8100 Hwy 71 S, Alexandria LA 71302 USA ([email protected]; [email protected]; [email protected]) This laboratory exercise introduces students to DNA extraction, biological barcoding, and sequence analysis; and reinforces the concept of the polymerase chain reaction (PCR). It takes advantage of the dipstick extraction technique that is cheap and fast and allows for DNA extraction and PCR setup on the same day in an 1:50 hr Introductory Biology lab. Students determine whether local businesses actually use the fish that they advertise, or if cheaper fish are being passed off as the more expensive species in order to boost profits. Different species of fish are collected from local vendors; DNA from their tissues is extracted; and a region from the Cytochrome C Oxidase I gene is amplified. After PCR, results are sent off for sequencing. The following week, chromatogram results are uploaded to NCBI, they are compared to existing genomic datasets, and conclusions are discussed in class. First page Keywords: DNA Barcoding, Dipstick DNA Extraction, PCR, molecular techniques, taxonomy Introduction with a completely new, inexpensive, and easy to perform DNA extraction methodology, dubbed the dipstick Some molecular techniques, such as PCR and gel technique (Zou et al., 2017). Specifically, students study electrophoresis, are so essential to modern biological PCR, learn about DNA extraction, discover an exciting and research that they are introduced and integrated into practical field of research, and practice their analytical Introductory Biology courses and laboratory exercises. skills using DNA sequences. Extraction of DNA material and DNA barcoding are also In these exercises, students perform DNA important techniques with wide-ranging practical barcoding analyses on fish samples taken from various applications but are not often included in such classes due markets, restaurants, or other vendors in town. Their to lengthy procedures. However, with the advent of more objective is to determine if the DNA they extract from the expeditious extraction methods, these procedures are well- tissue samples belongs to the species from which the suited for use in introductory courses. The authors use businesses are advertising, or if it belongs to a species that these techniques in a two-part series. The first set of lab is cheaper to market and has been fraudulently passed off exercises focuses on introducing students to PCR and gel as the original. During the first week, students will extract electrophoresis, and the second set focuses on DNA DNA and amplify the barcoding region (Cytochrome C extraction and DNA barcoding. The latter is presented Oxidase I) using PCR. The PCR product will then be sent here. to a local sequencing facility for analysis. During the Although DNA barcoding has been used second week, they will learn to read their resulting previously in undergraduate courses (Met et al., 2013; chromatograms and will perform BLAST searches to Butler et al., 2014; Robinson and Spindler, 2015), it has identify their species. been focused toward taxonomy, biodiversity, and ecology, Though these exercises were initially designed for and those exercises utilized more expensive and lengthy use in a college-level Introductory Biology class for DNA extraction techniques which may leave some majors, they can be modified to work in nonmajors labs as institutions with time constraints or budgets that make their well as labs at the high school level, or even with summer use prohibitive. The goal of this project was to introduce science camps or related programs. these techniques to a majors Introductory Biology course © 2020 by Louisiana State University at Alexandria 1 Major Workshop: DNA Barcoding Student Outline Objectives • Describe multiple applications of DNA barcoding • Describe the methodology involved in DNA barcoding • Extract DNA from tissue samples • Perform PCR to amplify a specific region of DNA • Analyze DNA sequence data • Use online DNA barcoding database to identify an unknown species Prereading: Review the structure and function of DNA before coming to lab. Also, review the concepts of 1) DNA replication, 2) transcription, 3) translation, 4) electron transport chain (ETC), and 5) the process of PCR. A thorough understanding of the methodology of this lab will rely on mastery of those concepts. For a review of the PCR procedure, please study this video: https://www.dnalc.org/resources/animations/pcr.html Introduction DNA barcoding is a molecular technique used for identifying the species of an unknown individual based on its DNA. The barcoding process utilizes short segments of DNA that are present in all major groups of species. The genetic regions that are used for those groups are selected because they evolve rapidly enough that each species has accumulated enough mutation to have its own genetically distinct sequence, but not so rapidly that different individuals of the same species have too many differences to be grouped together. The genetic region used to barcode most animal species comes from a 648 base-pair segment of the cytochrome C oxidase (COI) gene. This gene is located in mitochondria, and it codes for the “I” (pronounced “one”) subunit of the cytochrome C protein. You are already familiar with COI, as it is the quaternary-structure protein complex at the end of the electron transport chain (ETC) of cellular respiration. That protein complex, called either cytochrome c oxidase, COI, or complex IV, is responsible for building the proton concentration gradient across the inner membrane, as well as transferring electrons from the ETC to O2 to form water. This region has been shown to be highly effective at identifying many different invertebrate and vertebrate animal groups, including fish. In fact, you will be using it to identify fish in this lab. The workflow for a standard DNA barcoding identification involves 4 steps (Fig. 1). Tissue samples from the specimen must be obtained and DNA extracted from the tissue. Targeted gene regions (COI if you are identifying an animal) must then be amplified using PCR, and then those gene regions must be sequenced. Lastly, the DNA sequences of the sample are compared to DNA sequences from known species. Data from known species are available through online repositories such as the National Center for Biotechnology Information (NCBI). The methodology is easy and reliable, and it Figure 1. Standard workflow for the identification of only requires a small sample of tissue to work. Most forms of visual an unknown species using DNA barcoding. identification require the entire organism. Because of these benefits, 2 Advances in Biology Laboratory Education Stumpf, Bowers, and Sammons DNA barcoding has numerous biological applications including verification of disease vectors, preserving endangered species, monitoring water quality, and authentication of product ingredients such as food and medicines. Fish Fraud in Louisiana In early 2018, enforcement agents from the Louisiana Department of Wildlife and Fisheries (LDWF) received a tip that a number of restaurants in central Louisiana were advertising “catfish” to their customers while actually selling Swai, which is a much cheaper fish native to Southeast Asia. On January 26th, after investigating the matter, LDWF agents cited Rosie Jo’s, Crazy Cajun, and Debarge’s Crawfish restaurants (all very prominent eateries in Alexandria, LA) for engaging in fraud (Fig. 2). Figure 2. KALB news story describing episodes of fish fraud uncovered in central Louisiana. In this lab, you will be investigating how widespread a problem fish fraud is in central Louisiana. Your instructor has collected a series of fish samples from local business across the area, and you will perform a DNA barcoding analysis on the samples to see if they are actually the species they are advertised as being. The first week of this experiment you will extract DNA from your fish samples, complete PCR amplification on the COI gene, and prepare the PCR products to be sent out for DNA sequencing. The second week you will examine your returned sequence data and use an online data repository to verify the identity of your unknown tissue sample. Week 1 Procedure 1: Preparing the PCR Tubes In this procedure you will prepare several PCR tubes with the solutions you will need to amplify your DNA during the PCR procedure. The benefit to preparing the tubes before you extract your sample DNA is that you can begin the PCR process quickly after your DNA is successfully extracted. For this procedure you will prepare 50 µL reactions in each tube. This means the total volume of all solutions adds up to 50 µL. See Table 1 for itemized list of what goes into each tube. Note that the ONETAQ master mix contains Taq Polymerase as well as dNTP’s and reaction buffers. You will prepare one PCR tube for each fish sample and an additional PCR tube for the negative control. There is no positive control in this particular experiment, but successful DNA amplifications can become future positive controls for PCR (positive control = experiment that has already worked well). Publication of the Association for Biology Laboratory Education, Volume 41, 2020 3 Major Workshop: DNA Barcoding Table 1. List of components and their corresponding volumes needed for PCR amplification. Components Volume Forward primer 3 µL Reverse primer 3 µL ONETAQ master mix 25 µL Molecular grade H2O 19 µL Extracted DNA from your sample Total 50 µL General Rules: 1. Always wear gloves; you want to amplify DNA material from the fish, not your own cells! 2. Note how many reactions you will prepare before you even begin 3. Keep water, ONETAQ master mix, and primers on ice at all times 4.